PROJECT ABSTRACT Zika virus (ZIKV) is a reemerging mosquito-borne flavivirus that presents a formidable health threat substantiated by neurological and developmental anomalies and a sexual transmission route. The significant knowledge gap, as well as the lack of antiviral therapies and vaccines, has greatly increased the urgency of ZIKV research. Based on the precedents set by the investigations of N6-methyladenosine (m6A) in HIV-1, HCV, and ZIKV, we hypothesize that RNA post-transcriptional modifications (PTMs) play important roles in ZIKV infection by regulating essential functions in viral gene expression in different hosts. Understanding these functions will reveal new promising targets for antiviral development. The presence of PTMs on the genome of various RNA viruses has been known for decades. A concerted approach combining immunoprecipitation of m6A-containing fragments and RNA-seq analysis has facilitated functional analysis of m6A. The lack of detection capabilities has however severely hindered such knowledge of the more than 140 other known PTMs. The development of a more versatile platform based on mass spectrometric (MS) analysis has allowed us to examine the global landscape of PTMs present in total RNA extracts of mock- and ZIKV-infected cells, as well as on viral genomic RNA isolated by affinity capture from infected cells and virions. Our exciting results have shown that the genome of ZIKV is decorated by 38 different types of PTMs other than m6A, and that these astonishing constellations present noticeable variations as a function of origin and conditions. In this proposal, we will initiate the functional study of viral PTMs by initially pursuing those with distinctive expression patterns in infected cells and virions. More specifically, we will use MS-sequencing to target different dimethyl-cytosine modifications that were prominent on intracellular ZIKV RNA, but not on packaged RNA. We will deplete the enzymes that install/remove dimethyl-cytosine modifications, mutate the ZIKV RNA to prevent PTM addition, and examine the biological impact on viral translation, replication, and assembly. We will perform these experiments with different viral strains and in different cell and mosquito lines. The results will provide unique insights into the impact of PTMs on virus-host interactions and gene expression during development. These insights will lay the groundwork for the development of novel antivirals targeting host factor essential for ZIKV infection, but also possible broad- spectrum antivirals active across all flaviviruses. This study will help establish the priorities and framework for the elucidation of the remaining viral PTMs, which will be pursued by dedicated project grants. Beyond the investigation of ZIKV biology, these principles and the enabling technologies developed in this R21 will be applicable to study other conditions involving RNA malfunctions, which will further substantiate the broad transformative impact of this project.